Do I have to introduce the start codon of my gene of interest when I clone it downstream of the signal peptide?
A.: Do not incorporate the start codon again, but start with the second codon of your gene of interest. For good processing by the signal peptidase the signal peptidase cleavage site (AGA) may be followed by an alanine (… AGAA…). Other aminoacids at the N-terminus are allowed. In Bacillus subtilis the following amino acids occur at this position in secreted proteins: A, Q, E, K, D,V, F, N, L, D, T (Antelmann et al., 2001). Below you see pMM1525 showing the signal peptide and the open reading frame with its MCS. Cloning in SfoI allows to choose the N-terminal amino acid freely. Cloning in Bgl II results in an alanine (A) at the N-terminus of the secreted protein.
